Materials and Reagents

1. 220 ml bottom opened plastic pot
   
2. Glass petri dish, 9 cm in diameter
   Note: not necessary sterilized
   
3. Sterilized 50 ml glass culture bottle with cap
   
4. 1 ml glass pipette
   Note: Polypropylene pipette tips tend to retain Root-knot nematode (RKN), hence use of such tips should be avoided.
   
5. Sterilized 24-well tissue culture plate (e.g., IWAKI, catalog number: 3820-024 )
   
6. RKN Meloidogyne incognita (NIAS Genebank, MAFF number: 108258 )
   Note: We obtained a strain 108258 of RKN from the MAFF gene bank of the National Institute of Agrobiological Sciences of Japan (http://www.gene.affrc.go.jp/about_en.php). This strain can survive and propagate on plants carrying Mi-genes for RKN resistance (resistance breaking strain).
   
7. Tomato (Solanum lycopersicum), cultivar Momotaro
   Note: The cultivar Momotaro carries Mi-genes for RKN resistance. Seeds of tomato cultivars possessing Mi-genes (e.g., Momotaro) can be obtained from a seed company (Takii & Co. Ltd., Kyoto, Japan).
   
8. Arabidopsis thaliana (Columbia)
   
9. Water (Milli-Q grade)
   
10. Sodium hypochlorite solution (Nacalai Tesque, catalog number: 31518-35 )
    
11. Ethanol (99.5%) (Nacalai Tesque, catalog number: 09666-85 )
    
12. Sea sand with an average size of 0.3 mm
    Note: Shift and wash sea sand to remove the pebbles and the salt with a large amount of tap water in a metal tub and autoclave the washed sand.
    
13. Liquid fertilizer (Hyponex)
    
14. Methanol (99.8%) (Nacalai Tesque, catalog number: 21915-93 )
    
15. Acid fuchsin (e.g., Sigma-Aldrich, catalog number: F8129-25G , or Wako, catalog number: 061-01332 )
    
16. Lactic acid (85~92%) (Nacalai Tesque, catalog number: 20006-75 )
    
17. Glycerol (99%) (Nacalai Tesque, catalog number: 17018-83 )
    
18. Sclareol (Sigma-Aldrich, catalog number: 357995-1G )
    
19. Sodium hydroxide (Nacalai Tesque, catalog number: 31511-05 )
    
20. Acetic acid (99%) (Nacalai Tesque, catalog number: 00211-95 )
    
21. Acid fuchsin solution (see Recipes)
    
22. 100 mM sclareol solution (see Recipes)

Equipment

1. Greenhouse or chamber for plant growth
   
2. Stainless tweezer
   
3. Incubator (e.g., Sibata, model: SMU-0541 )
   
4. Light microscope (e.g., OLYMPUS, model: BX53M )
   
5. Refrigerator (e.g., PANASONIC, model: MPR-162DCN )
   
6. Stainless steel tray (9.2 cm in height x 18 cm depth x 32 cm width)
   
7. Scissors (e.g., V. Mueller, catalog number: 25601-164 )
   
8. Water bath (e.g., Taitec, model: SH-10N )
   Note: You can also use a commercially available microwave oven for cooking.
   
9. Microwave oven (e.g., HITACHI, model: MRO-NT5 )
   
10. Stereoscopic microscope (e.g., Nikon, model: SMZ-U )

Procedure